Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I agree with Leoncio here - we have several fluorescence microscopes and
they all have monochrome cameras (of various sorts!) on them. The only
time a "true" colour camera is potentitalli useful is when you have a
filter system to view multiple labels simultaneously. But while they can
produce a pretty picture sometimes, we have a cupboard full of them that
are never used in practice... in the end, people usually need to know
what each label is showing them in isolation from the others... which
leads to the point: a single labelling channel has only "monochrome"
information: red, green or whatever colour dyes like Cy5 or Cy7 really
are...

IAN


"Vergara, Leoncio A." wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Sorry, I must apologize for the tone of the last sentence in my message. I
> understand the opinion of the "pro-color" investigator, but I don't agree.
> This is an old issue, as the director of a multi-user facility I must deal
> with it all the time and I am a bit tired of giving the same explanation
> over an over.
> I have the impression that your investigator is in the spirit of color film
> photography and doesn't understand the benefits of digital imaging.
>
> One funny aspect of this discussion is that if you analyze the way color
> cameras work, you realize that they do automatically exactly the same that
> you do when taking multiple monochrome images and combine them by software,
> it is all pseudocoloring at the end!
>
> Although you can balance the exposure times for the different RGB components
> in a color camera, you have more flexibility when using separate filters to
> compensate for different brightness of multiple stains, change exposure
> time, add or remove neutral density filters, etc.
>
> With monochrome cameras you can benefit from high quality narrow band
> filters for accurate dye separation and deal with issues like bleed-trough.
> Quantification is another issue tied to dye separation.
>
> However, I can see some practical advantages of color cameras:
>
> 1 You can use the same camera for color bright field.
> 2 You can preview and focus in color
> 3 You can use those double and triple (and quadruple) filter sets, (what you
> see is what you get, right?).
>
> In our systems we always use monochrome cameras for fluorescence period. In
> one system we added a color camera for bright field (we can use a lower cost
> one because sensitivity is not as much of an issue). In another system we
> added an emission filter wheel that contains an RGB filter set and used an
> automatic acquisition sequence for Metamorph, all with the idea of
> satisfying those hard-headed users...
>
> Leoncio Vergara
>
> -----Original Message-----
> From: ray hester [mailto:[log in to unmask]]
> Sent: Friday, May 24, 2002 2:35 PM
> To: [log in to unmask]
> Subject: monochrome vs color CCD camera
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I had assumed that it would be better to buy a black/white cooled CCD camera
> to be used for the acquisition of images (DAPI/FITC/TRITC/etc.) on our
> fluorescence microscope (with 'colorization' assigned post-acquisition with
> software), but an investigator here has continued to insist that we need to
> buy a color CCD camera.  Some of his comments follow.
>
> I would very much appreciate any input as to which type of camera
> (monochrome vs color) those of you who have this set-up (cooled CCD camera
> on a fluorescence scope) are using for acquisition of fluorochromes such as
> those mentioned above.
>
> Thanks in advance.
>
> Ray Hester
> Univ. of South Alabama
> Mobile, AL 36688
> [log in to unmask]
>
> ______________________________
>
> Comments of pro-color investigator:
>
> I must admit that I'm a bit dumbfounded about the rationale for buying a
> monochrome camera. This does not appear to be the general recommendation for
> imaging immunofluorescence at large meetings like Neuroscience. Perhaps the
> advice reflects the more skewed applications of some of our users. We have a
> monochrome option on our SPOT camera which does indeed require much shorter
> exposures than color. But we find that pseudocoloring produces overly
> contrasty images which are acceptible for  some applications, e.g.,
> pseudocoloring darkfield in situ hybridization images, but impart an
> artifactually high intensity of labeling for immunocytochemistry. With
> respect to immunofluorescence I balk at manipulating data (pixels) like
> this, and as a reviewer am troubled when I see it.

--
Professor Ian Gibbins
Anatomy & Histology
Flinders University of South Australia
GPO Box 2100, Adelaide, SA 5001
Australia

Phone:  +61-8-8204 5271
FAX:    +61-8-8277 0085
Email:  [log in to unmask]